We continue to develop our biophysical measurement systems (biological atomic force microscopy (Bio-AFM) platforms, Quartz Crystal Microbalance-Dissipation (QCM-D), and optical microscopy and spectroscopy), and to apply these technologies to important biomedical investigations in collaboration with outstanding NIH intramural and extramural scientists. On the instrumentation front, we have built a new high-speed AFM from home-made and open-source components with scan rates up to twenty lines per second. We are continuing an integration of Raman spectroscopy with Bio-AFM, and working toward broader applicability of AFM imaging and single molecule force spectroscopy (SMFS) for cellular and bio-molecular sample characterizations. On the application front, our major collaborations and notable results in this year include: (1) We have maintained our commitment to work on clinical vaccine development toward enhanced immunological response and eventual eradication of malaria. We have investigated the macromolecular structure and nanomechanical properties of more malaria vaccine candidates and virus-like-particle carriers via Bio-AFM and related bioanalysis with Dr. David Narum (Laboratory of Malaria Immunology and Vaccinology, NIAID, NIH), and other collaborators. These malaria protein antigens and vaccine carriers are produced via recombinant-protein biotechnology, purified, and characterized in a manner suitable for human trials and scale-up production. Biophysical characterization at single macromolecule and assembly level using Bio-AFM imaging and force spectroscopy are helping define these vaccine constructs along the developmental phases. We also aim to improve mechanistic understanding of the malaria parasites and pathogen-host interactions. (2) We have expanded our collaboration on multifunctional nanomedicine probes with Dr. Xiaoyuan Chen (laboratory of Molecular Imaging and Nanomedicine, NIBIB), Dr. Ashwin Bhirde, Dr. Peng Huang Dr. Dingbin Liu, and an international team of co-investigators. Dye-loaded ferritin nanocages, RNA-nanoparticle and carbon nanotube/graphene oxide based theranostics are among nanomedicine, Bioimaging and biomarker detection probes studied via Bio-AFM and published this year. Toward multifunctional cellular imaging and biomedical applications, we are examining broader applications of Bio-AFM and QCM-D methodology for investigating nanoparticle theranostics and their impact on cancer cells, stem cells, and related biomedical systems. (3) We have continued our Bio-AFM studies of protein clathrin and assemblies with collaborators including Drs. Ralph Nossal and Dan Sackett(NICHD, NIH), and Prof. Eileen Lafer (Univ. Texas Health Sciences Center, San Antonio). Clathrin is a key protein for receptor-mediated endocytosis and intracellular trafficking. Further Bio-AFM and QCM-D measurements have been pursued this year to characterize clathrin and its assembled structures, as well as interaction with several partner proteins important to the function of cells. We published with Dr. Ling-gang Wu (NINDS) and coworkers on membrane structural changes in exocytosis and endocytosis of dense-core vesicles and their implications for synaptic transmission in brain. (4) We have collaborated with NIDCR scientists, Drs. Andrew Doyle and Kenneth Yamada (Laboratory of Cell and Developmental Biology, NIDCR), on nanomechanics and structural properties of tissue specific extracellular matrices and reconstituted matrix-like gels via Bio-AFM force spectroscopy. Collaborating also with Dr. Raimon Sunyer (NICHD and currently at Institute for Bioengineering of Catalonia, Spain), we are defining the best force spectroscopy approaches to explore tissue-mimicking gel matrices and their relevance to cell adhesion and migration. (5) Among other continuing collaborations this year, we have investigated further protein and DNA interactionnns and nucleosome structure and dynamics, critically important in gene regulation and disease mechanisms, with Dr. Yawen Bai (Laboratory of Molecular Cell Biology, NCI) and coworkers. We have collaborated with Prof. Xiangyun Qiu (George Washington University) and his group on DNA cross-linking and related studies. And with other groups of collaborators (e.g. Dr. Richard Hendler, NHLBI), we have advanced our Bio-AFM and biophysical studies of several protein fiber assemblies, such as amyloid-beta fibers implicated in the Alzheimer's disease. (6) We have initiated new collaborative projects this year. We have started to collaborate on an INBRE pilot project with Drs. Qi Lu, Hacene Boukari and Yuriy Markushin on the effects of nanoparticles on the organization of lipid domains toward cancer prevention and treatment. We have started to work with Dr. Yun-Xing Wang (NCI) and colleagues on determining high-resolution structures of a key human immunodeficiency virus(HIV) RNA and its complex with co-factors and developing therapeutics.